215
Advances in Genome Editing Tools
Prime-editing is a related method that allows slightly larger
changes to be made using Cas9 nickase fused to reverse
transcriptase and a complex multifunctional prime-editing
sgRNA (Anzalone et al., 2020). At the time of writing, this
technique is to our knowledge yet to be used in Xenopus
with success.
14.5. CONCLUSIONS
The range of methods currently available to Xenopus
researchers for effcient genome modifcation is extensive
and contains robust techniques for both transgenesis and
mutagenesis. When combined and used together with other
experimental manipulations commonly used in Xenopus,
such as tissue-targeted microinjection, for example, they
permit the pursuit of experimental questions not easily
explored in other model systems. Mutations in human
PKD1 are associated with autosomal-dominant polycystic
kidney disease (ADPKD), and homozygous mutations or
deletions of pkd1 in mice are embryonic lethal (Blackburn
and Miller, 2019). Similarly, CRISPR-Cas9 induced mutations in X. laevis pkd1 result in F0 edema and eventual
lethality (Figure 14.1A). To bypass the embryonic lethality and more specifcally target the germline for generation
of mutant lines, pkd1 sgRNAs can be microinjected into
the vegetal pole at the 16-cell stage (Figure 14.1B). The
effects of pkd1 mutations on early kidney morphogenesis
can, however, be easily observed in Xenopus if induced in
the pax8:GFP transgenic background in which the pronephros is labeled with GFP. This is even more powerful
if combined with unilateral mutagenesis where the pkd1
sgRNA injected side shows disrupted pronephric morphology, and the other side injected with a control sgRNA provides a morphologically normal internal control (Figure
14.1C,D ). Xenopus, which has always been a powerful system in many respects, has bravely leapt into the heart of
the genetic age.
ACKNOWLEDGMENTS
We wish to thank Ira Blitz for providing helpful comments
during the writing of this chapter. We apologize to those
individuals whose publications we missed in compiling this
FIGURE 14.1 CRISPR knockout of pkd1 in X. laevis. (A) Ten-day-old F0 pkd1 knockout tadpole with severe edema; these tadpoles die
before 14 days. (B) Six-month-old F0 pkd1 frogs generated by injecting the sgRNA into vegetal blastomeres at the 16-cell stage. These F0
tadpoles do not generate edema and survive to adulthood. (C) Left side of Xla.Tg(pax8:GFP) Ogino transgenic tadpole injected with pkd1
sgRNA. Pronephric tubules are dilated. (D) Uninjected right side of tadpole showing normal pronephric tubules.
Advances in Genome Editing Tools
Prime-editing is a related method that allows slightly larger
changes to be made using Cas9 nickase fused to reverse
transcriptase and a complex multifunctional prime-editing
sgRNA (Anzalone et al., 2020). At the time of writing, this
technique is to our knowledge yet to be used in Xenopus
with success.
14.5. CONCLUSIONS
The range of methods currently available to Xenopus
researchers for effcient genome modifcation is extensive
and contains robust techniques for both transgenesis and
mutagenesis. When combined and used together with other
experimental manipulations commonly used in Xenopus,
such as tissue-targeted microinjection, for example, they
permit the pursuit of experimental questions not easily
explored in other model systems. Mutations in human
PKD1 are associated with autosomal-dominant polycystic
kidney disease (ADPKD), and homozygous mutations or
deletions of pkd1 in mice are embryonic lethal (Blackburn
and Miller, 2019). Similarly, CRISPR-Cas9 induced mutations in X. laevis pkd1 result in F0 edema and eventual
lethality (Figure 14.1A). To bypass the embryonic lethality and more specifcally target the germline for generation
of mutant lines, pkd1 sgRNAs can be microinjected into
the vegetal pole at the 16-cell stage (Figure 14.1B). The
effects of pkd1 mutations on early kidney morphogenesis
can, however, be easily observed in Xenopus if induced in
the pax8:GFP transgenic background in which the pronephros is labeled with GFP. This is even more powerful
if combined with unilateral mutagenesis where the pkd1
sgRNA injected side shows disrupted pronephric morphology, and the other side injected with a control sgRNA provides a morphologically normal internal control (Figure
14.1C,D ). Xenopus, which has always been a powerful system in many respects, has bravely leapt into the heart of
the genetic age.
ACKNOWLEDGMENTS
We wish to thank Ira Blitz for providing helpful comments
during the writing of this chapter. We apologize to those
individuals whose publications we missed in compiling this
FIGURE 14.1 CRISPR knockout of pkd1 in X. laevis. (A) Ten-day-old F0 pkd1 knockout tadpole with severe edema; these tadpoles die
before 14 days. (B) Six-month-old F0 pkd1 frogs generated by injecting the sgRNA into vegetal blastomeres at the 16-cell stage. These F0
tadpoles do not generate edema and survive to adulthood. (C) Left side of Xla.Tg(pax8:GFP) Ogino transgenic tadpole injected with pkd1
sgRNA. Pronephric tubules are dilated. (D) Uninjected right side of tadpole showing normal pronephric tubules.